Who Is It For?

Lecture 32 Flashcards Preview

Sterile mutants of yeast)

- Lack of cell surface receptors- Which make up a heterochromatic complex- They interact with the pheromones, and if there are mutations within this complex, it will not be able to recognise other pheromones, so the phosphorylation cascade will not be activated

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Ligand binding and GTPases:

- Exchange of GDP and GTP is a good way to turn proteins (especially GTP proteins) off and on)- Intrinsic GTP activity is low without an effector protein, ( they are slow to hydrolyse the GT and add a phosphate)- GDP is active, GAP activates it, so it increases its GTPase activity, hydrolyses it's domain and is now inactive.- Liberate GDP from it's inactive form using guanine nucleotide dissociation inhibitors (GDI)- Regulation of gene expression at the protein level by exchanging GTP and GDP

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Protein processing:

- Specifically cleaving proteins to give it different activity- Specific proteases result in different polypeptide segments which can do different things compared to what it would do if it was cleaved by a different set of proteases

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Translational control:

- Before you make the protein, so controls the timing of protein production- Can occur at multiple levels- Destabilising - Changing when/if translation starts

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mTor Kinase:

- A major regulatory kinase of transitional state- mTor (mouse Tor kinase) controls when a particular mRNA is translated- If a particular growth factor is present TorK will phosphorylate 4E-BP- 4E-BP in the phosphorylated state cannot bind the initiation complex required to recruit other elongation factors and finally the ribosome, translation will occur!- When 4E-BP is not phosphorylated it can bind, and the elongation factor 4E is kicked out, so it stops the formation of the ribosomal machinery, so no translation occurs

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Iron regulatory protein:

- Iron is required for iron kelators in the body, but too much is toxic, so it must be regulated- an element can form a secondary structure called the iron regulatory element (a loop in mRNA)- IRP will bind this secondary structure, preventing the ribosomal machinery assembling, so not translation occurs- When iron levels increase, it binds to IRP so that it cannot bind the loop so it no longer inhibits the translational machinery, so transcripts are translated

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Post transcriptional control:

mRNA stability in RBC:

- RBC don't have nuclei, so the RNA for RBC proteins comes from a progenitor- RNA encoding hemoglobin must be stable so that they can be translated many times, without needing to replace it- Stabilisation occurs because of the cell type the mRNA is deposited in

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mRNA stability in aspergillus nidulans:

- Ammonium can be sourced from the environment, but can also be expressed from the genome- A transcriptional activator encoded by the areA gene is involved in positively acting to transcriptionally regulate genes involve din assimilating nitrogen sources other than ammonium. - IT binds to the promoters of genes in the absence of ammonium, by creating an mRNA which is translated- This mRNA has differentially stability based on the presence (10 mins half life) or absence (40 mins) of ammonium- This difference in timing result due to the binding of de-adenylases on the 5' polyA tail, which gets shorter and shorter and hence more unstable

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RNA localisation in saccaromyces cerivisiae:

- Mother cells can switch cell type- If the mother creates two daughter cells, the bigger one is able to switch mating types- This switching process occurs to HO, homothallic - a cell that can mate with itself, HO is dominant over Ho (must mate with the opposite mating type)- If Ho gets turned off it can't switch mating types- Swi5: found in both mother and daughter cell types - Ash1: only found the in daughter, not the mother. Ash1 is only translated in the daughter nucleus

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Post-transcriptional control:

- RNA processing including alternate splicing

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Alternate splicing - negative control:

- A primary transcript may splice an intron out in normal conditions- In a different condition a protein may bind the intron/exon boundary, blocking the machinery from correctly splicing. A protein with a different function may result, or a nonsense protein may occur

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Alternate splicing - positive control:

- The primary transcript will usually leave the intron in and so there is no function or it has a particular action- An activator protein that binds the RNA helps splicing occur properly, so that a different/functional protein is created by inducing splicing

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Dosage compensation in sex determination of drosophila:

- In mammals, everyone has one lot of X chromosome genes, by inactivating one X chromosome in females- In flies, the X chromosome in males is upregulated (males are X, females are XX)- Sex is determined by the ratio of X chromosomes to autosomes. - Splicing at the sex lethal locus occurs due to having 2 X's. This leads to female development.- Only one X chromosome so turns off splicing in sex lethal, turning off the transformer gene. This leads to male development- This is all post-transcriptional!

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The ratio of X to autosomes determines what happens at the sex lethal locus (males):

- Sxl gene, Tra gene, Dsx gene, male or female.FOR A MALE:- In a male splicing occurs at the Sex lethal locus, producing a nonfunctional protein product- The transformer gene produces a non-functional productdue to splicing- The double sex gene is spliced from the 5' of intron 1 to the 3' of intron 2- This is translated to produce a male-specific amino acid- This repressed female differentiation genes and male development genes

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The ratio of X to autosomes determines what happens at the sex lethal locus (females):

FOR A FEMALE:- The ratio of X chromosomes determines the level of sex lethal proteins- A high level of this protein will block the splice site in the sex lethal protein, so it is a self-regulating system- The sex lethal product binds to the 1st splice site of the transformer gene, so a functional transformer protein is produced- Differential splicing occurs in double sex due to the binding of the transformer protein- The double sex protein represses male differentiation genes and leads to female development

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DNA modification:

- Usually only occurs at CpG doublets- In humans there is a lot of methylation. In drosophila there is almost no methylation. Methylation maybe isn't the primary way of controlling gene expression. The effects are gene and organism dependent

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Hw does methylation operate?

- There are two classes of DNA methylases- De novo methylase (unmehtylated DNA can be methylated) and maintenance methylase (maintain methylation after DNA replication)- During replication the 2 strands separate, so one strand is methylated (it is a hemimethylated ds strand), but the matching G is not. Re-establishing methylation requires maintenance methylase to completely methylate the double strand- Not maintaining methylation results in loss of methylation after replication-

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DNA demethylase:

- Does the opposite of de novo methylase, it takes methylase groups off

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How do DNA methylases act?

- Co-valent changes on the DNA don't in themselves do anything.- They recruit proteins that can change the expression patterns of the strand they bind- DNA methylation can result in silencing of gene expression- Effect recruitment of histone modifying enzymes